Research Article
3897
The Clp1p/Flp1p phosphatase ensures completion of
cytokinesis in response to minor perturbation of the
cell division machinery in Schizosaccharomyces
pombe
Mithilesh Mishra1,*, Jim Karagiannis1,*, Susanne Trautmann2, Hongyan Wang1, Dannel McCollum2 and
Mohan K. Balasubramanian1,3,‡
1Cell Division Laboratory, Temasek Life Sciences Laboratory and 3Department of Biological Sciences, National University of Singapore,
1 Research Link, NUS, Singapore 117604, Rep. of Singapore
2Department of Microbiology and Molecular Genetics, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 016550122, USA
*These authors contributed equally to this work
‡Author for correspondence (e-mail: [email protected])
Accepted 5 April 2004
Journal of Cell Science 117, 3897-3910 Published by The Company of Biologists 2004
doi:10.1242/jcs.01244
Summary
Fission yeast mutants defective in actomyosin ring
formation and function exhibit a prolonged G2 delay
following cytokinesis failure. This G2 delay depends on the
SIN, a signaling network essential for cytokinesis, and the
non-essential Cdc14p family phosphatase, Clp1p/Flp1p
and has been proposed to signify a cytokinesis checkpoint
mechanism. However, the physiological relevance of this
proposed Clp1p/Flp1p-dependent checkpoint is unclear
because all previous studies were carried out using
mutations in essential actomyosin ring components under
fully restrictive conditions and thus these cells would have
died regardless of the presence of the checkpoint. Here
we show that delays in cytokinesis caused by minor
perturbations to different components of the cytokinetic
machinery, which normally cause only mild defects,
Introduction
The production of two viable daughter cells in eukaryotes
depends on the execution of DNA synthesis, mitosis and
cytokinesis in a strict sequential order. Thus, mitosis is initiated
only upon completion of DNA synthesis and cytokinesis is
initiated only upon entry into anaphase. In addition, checkpoint
mechanisms operate to ensure that DNA is not damaged before
entry into mitosis, and that kinetochores have attached to the
mitotic spindle before anaphase onset. The mechanisms that
ensure alternation between S and M phases, and those that
ensure that DNA is not damaged and is attached to the mitotic
spindle, operate to produce genetically identical daughter cells
that are viable (Kelly and Brown, 2000; Nyberg et al., 2002;
Rudner and Murray, 1996). A key question in the regulation of
cytokinesis relates to the nature of checkpoint mechanisms that
might monitor the completion of this process.
Cytokinesis is the stage in the cell cycle where barriers
between divided nuclei are assembled and involves the
function of an actomyosin-based contractile ring in several
eukaryotes (Field et al., 1999; Gould and Simanis, 1997;
become lethal when Clp1p/Flp1p is inactivated. In
addition, we show that Clp1p/Flp1p does not function
simply to inhibit further rounds of nuclear division, but
also allows damaged actomyosin rings to be maintained to
facilitate completion of cell division. Ectopic activation
of the SIN significantly bypasses the requirement of
Clp1p/Flp1p for G2 delay as well as for completion of
cytokinesis. We conclude that the Clp1p/Flp1p-dependent
cytokinesis checkpoint provides a previously unrecognized
cell survival advantage when the cell division apparatus is
mildly perturbed.
Movies available online
Key words: Fission yeast, Cytokinesis, Checkpoint
Guertin et al., 2002; Feierbach and Chang, 2001). The fission
yeast, Schizosaccharomyces pombe, is an attractive organism
for the study of cytokinesis, because its cell cycle is well
characterized and because this yeast divides using an
actomyosin-based ring. In addition, a large bank of mutants
defective in cytokinesis have been identified in this yeast
(Nurse et al., 1976; Balasubramanian et al., 1998; Chang et al.,
1996; Schmidt et al., 1997).
Cytokinesis mutants in fission yeast (Nurse et al., 1976;
Balasubramanian et al., 1998; Chang et al., 1996; Schmidt et
al., 1997) can be divided into two broad categories (Table 1).
Group I comprises those in which mitotic cell cycle
progression is delayed upon cytokinetic failure (Liu et al.,
1999; Le Goff et al., 1999b; Trautmann et al., 2001; Cuellie et
al., 2001; Nurse et al., 1976; Liu et al., 2000). Actomyosin ring
assembly and cell wall synthesis mutants fall under this
category. Group II comprises those in which the timing of
mitotic cell cycle progression is unperturbed following
cytokinetic failure (Nurse et al., 1976; Liu et al., 2000; Le Goff
et al., 1999b). The SIN (septation initiation network) mutants,
3898
Journal of Cell Science 117 (17)
Table 1. Cytokinesis mutants
Group I
Group II
cdc3
cdc4
cdc8
cdc12
cdc15
act1
rng2
rng3
rng5/myo2
cps1
cdc7
cdc11
cdc14
sid1
sid2
sid3/spg1
sid4
mob1
which define components of the spindle pole body that regulate
the timing of cytokinesis (Le Goff et al., 1999a; McCollum and
Gould, 2001), belong to this category. Interestingly the mitotic
delay following failed cytokinesis in group I mutants is
abolished when combined with SIN mutations, suggesting that
SIN function is important for mitotic delay, as well as proper
cytokinesis (Liu et al., 2000; Le Goff et al., 1999b). Loss of
function mutations in the protein phosphatase Clp1p/Flp1p
(hereafter referred to as Clp1p) also allow bypass of the mitotic
delay in group I mutants (Trautmann et al., 2001; Cuellie et al.,
2001). Thus, Clp1p, as with the SIN, appears to play a role in
the mitotic delay, but unlike the SIN is not essential for
cytokinesis.
The ability of group I mutants (in essential actomyosin ring
assembly and cell wall synthetic enzymes) to delay G2/M
progression in response to failed cytokinesis implicates a
Clp1p-dependent checkpoint mechanism that monitors
cytokinesis (Liu et al., 2000; Le Goff et al., 1999b; Trautmann
et al., 2001; Cueille et al., 2001). The physiological
significance of this Clp1p-dependent G2 delay mechanism,
however, was unclear because, unlike in previously described
checkpoints, mutant cells died regardless of the presence of the
checkpoint.
In this study we show that the Clp1p-dependent cytokinesis
checkpoint provides a previously unrecognized cell survival
advantage. We show that Clp1p, normally a non-essential
protein, becomes critical for maintaining viability upon mild
perturbation of the cytokinetic machinery. Furthermore, we
provide evidence that Clp1p acts in two ways to aid survival.
It is required both for the maintenance of cell division
structures upon damage, as well as halting the nuclear cycle.
In addition, we demonstrate that ectopic signaling from the
SIN compensates for loss of Clp1p in conditions where the
cytokinetic machinery is damaged. Given that cell division
structures similar to those seen in S. pombe are observed in
more complex eukaryotes (Guertin et al., 2002), and that Clp1p
(as well as some components of the SIN) are conserved
(Bembenek and Yu, 2001; Gruneberg et al., 2002; Moreno et
al., 2001; Gromley et al., 2003), a similar checkpoint
mechanism might operate in other organisms.
Materials and Methods
Schizosaccharomyces pombe strains, media and growth
conditions
The S. pombe strains used in this study are: leu1-32 ura4-D18 (wild
type), clp1∆ (Trautmann et al., 2001; Cueille et al., 2001), cps1-191
(Liu et al., 1999; Le Goff et al., 1999b), cdc11-123, cdc14-118,
cdc7-24, cdc15-140, cdc4-8, cdc25-22, cdc3-124, cdc12-112 (Nurse
et al., 1976), cdc16-116 (Minet et al., 1979), myo2-E1
(Balasubramanian et al., 1998), rng2-D5 (Eng et al., 1998),
myp2::his7+ (Bezanilla et al., 1997; Motegi et al., 1997),
rlc1::ura4+ (Naqvi et al., 2000; Le Goff et al., 2000), cyk3::ura4+
(W.H.Y. and M.K.B., unpublished), myo52::ura4+ (Win et al.,
2001), cdc14-118 myo2-E1, cdc11-123 myo2-E1, cps1-191 clp1∆,
cdc4-8 clp1∆, cdc15-140 clp1∆, cdc11-123 clp1∆, cdc3-124 clp1∆,
cdc12-112 clp1∆, myo2-E1 clp1∆, rng2-D5 clp1∆, cdc25-22 clp1∆,
cps1-191 cdc16-116, cdc16-116 clp1∆, cdc16-116 cps1-191 clp1∆,
myp2∆ clp1∆, rlc1∆ clp1∆, cyk3∆ clp1∆, myo52∆ clp1∆, rlc1gfp,
rlc1gfp clp1∆, rlc1gfp sid1-123, rlc1gfp sid2-250, rlc1gfp cdc7-24,
rlc1gfp cdc14-118. Cell culture and maintenance were carried out
using standard techniques (Moreno et al., 1991). Vegetative cells
were grown in YES medium. Genetic crosses were performed by
mixing appropriate strains of opposing mating type on YPD plates
and recombinant strains were selected by tetrad dissection carried
out on Singer MSM Micromanipulator (Singer instruments, UK).
Double mutants were typically isolated from NPD tetrads.
Elutriation was performed using a Beckman elutriation chamber (JE
5.0) according to the manufacturer’s instructions. Latrunculin A was
purchased from Molecular Probes (Eugene, OR). Lat A was used at
0.2 µM (in DMSO).
Fluorescence and time-lapse microscopy
Cell staining with 4′6,-diamidino-2-phenylindole (DAPI), aniline
blue, and rhodamine-conjugated phalloidin were performed as
described previously (Balasubramanian et al., 1997). Aniline blue
was used at a final concentration of 0.5 µg/ml. Aniline blue and
rhodamine-conjugated phalloidin was purchased from Sigma (St
Louis, MO). Images were acquired using a Leica DMLB
microscope in conjunction with an Optronics DEI-750T cooled
CCD camera and Leica QWIN software. Images were processed
and assembled using Adobe Photoshop 5.5. In experiments
involving quantification, at least 500 cells were counted. Timelapse microscopic studies were performed as previously described
(Wong et al., 2002). Briefly, S. pombe cells were grown in rich
medium until mid-exponential phase and then shifted to medium
containing 0.2 µM Lat A or DMSO (solvent control) for 30
minutes. 1 µl of cell culture was then mounted on borosilicate glass
slide covered with a coverslip (both from Matsunami Trading,
Japan). All time lapse microscopy images were obtained using a
Lieca DMIRE2 microscope equipped with Uniblitz shutter and
CoolSnap HQ CCD camera (Photometrics) driven by MetaMorph
4.6r9 software (Universal Imaging). Experiments were carried out
at approximately 25°C. In experiments involving temperaturesensitive SIN mutants, cells were grown in rich medium at 25°C
and shifted to the restrictive temperature of 36°C before being
mounted on a glass slide and imaged using a temperature-controlled
stage (MC 60 Linkman Scientific Instruments, UK) maintained at
36°C. At least 10 cells were imaged in all cases. 3D reconstructions
were performed by taking Z-series (100 nm sections) of wide-field
fluorescent images and de-convolving using the AutoDeblur/
AutoVisualize 9.2 package (Autoquant Imaging). Long-term
observation of histone-GFP expressing cells was performed using
a temperature-controlled flow chamber system (Warner Instrument)
filled with YES and maintained at 32°C as previously described
(Karagiannis and Young, 2001). Instead of poly-L-lysine, cells
were kept immobilized with the use of a small (600 mm diameter)
cylindrically shaped 0.8% agarose pad made in YES. The agarose
pad was made to the same height as the chamber and thus cells were
gently trapped between the agarose surface and the cover-slip
forming the bottom of the chamber. Cells were prepared for
immunofluorescence as previously described (Balasubramanian et
al., 1997). Rabbit anti-GFP primary antibodies (Molecular Probes
A-6455, Eugene, OR) and goat anti-rabbit IgG secondary
Cytokinesis checkpoint in fission yeast
antibodies (Molecular Probes A-11008), Eugene, OR) were used at
1:1000 and 1:800 dilutions, respectively.
Results
Clp1p is essential when the apparatus of cytokinesis is
mildly perturbed via mutations or chemical inhibitors
To evaluate the physiological function of Clp1p in coordinating
mitosis and cytokinesis, we made double mutants lacking
Clp1p and one of several components listed under group I in
Table 1. The chosen mutants included myosin light chain cdc48 (McCollum et al., 1995), myosin II heavy chain myo2-E1
(Balasubramanian et al., 1998), IQGAP-related molecule rng2D5 (Eng et al., 1998), PCH and SH3 domain protein cdc15140 (Fankhauser et al., 1995) and 1,3-β-glucan synthase cps1191 (Liu et al., 1999). We also made double mutants between
clp1∆ and four non-essential components of the actomyosin
ring, such as the type II myosin heavy chain Myp2p (Bezanilla
et al., 1997; Motegi et al., 1997), myosin regulatory light chain
Rlc1p (Naqvi et al., 2000; Le Goff et al., 2000), the SH3
domain protein Cyk3p (H.W. and M.K.B., unpublished), and
the type V myosin Myo52p (Win et al., 2001; Motegi et al.,
2001). Strikingly, we found that under conditions in which
single mutants containing wild-type clp1+ were able to divide
and form colonies, albeit with altered morphology, the double
mutants that lack Clp1p were inviable (Fig. 1A, and data not
shown). Single mutants containing clp1+ could assemble
functional septa (shown with arrowheads in Fig. 1B),
indicating successful cytokinesis, and rarely accumulated more
than two nuclei (Fig. 1C). However, when clp1 was deleted,
the double mutants, only infrequently assembled division septa
(Fig. 1B and C; data not shown) and a significant proportion
of double-mutant cells accumulated 4 or more nuclei (Fig. 1C).
Taken together these studies suggest that clp1∆ cells are
supersensitive to a wide variety of perturbations to the cell
division machinery, including mutations that affect assembly
of the actomyosin ring, division septum and secretion.
The importance of Clp1p in responding to minor damage of
cell division structures was further supported by our analysis
of wild-type and clp1∆ cells treated with a low dose of
latrunculin A (Lat A), a drug that prevents actin polymerization
(Ayscough et al., 1997). Typically, treatment of wild-type cells
with 10 µM or higher concentrations (high dose), leads to the
loss of all detectable F-actin structures and lethality (Pelham
and Chang, 2001). In contrast, 0.2 µM Lat A (referred to as
‘low dose’ in this manuscript) did not cause any detectable
defects in the appearance of F-actin structures in wild-type
cells although, the effect on F-actin function was detected as a
delay in progression through cytokinesis.
Wild-type and clp1∆ cells were synchronized in early G2 by
centrifugal elutriation, treated with low doses of Lat A, and
subsequently sampled at 30 minute intervals over a 6 hour time
frame. The kinetics of the nuclear cycle progression and
septation were then examined by staining with aniline blue and
DAPI, to visualize the division septum and nuclei, respectively
(Fig. 1D and E). Wild-type and clp1∆ cells treated with DMSO
showed similar degrees of synchrony with respect to mitosis
and septation. However, these strains differed dramatically
when exposed to low doses of Lat A. Although wild-type
and clp1∆ cells entered mitosis synchronously and with
comparable kinetics upon low-dose Lat A treatment, wild-type
3899
cells persisted as binucleates that slowly completed assembly
of the division septum that was abnormal in appearance, but
that completely bisected the cell (Fig. 1E; marked with
arrowhead). In contrast, clp1∆ cells treated with Lat A did not
assemble detectable division septa, exited mitosis with normal
kinetics, and completed another round of mitosis within the
time-frame of the experiment leading to the accumulation of
tetranucleate cells (Fig. 1D and E). Furthermore, wild-type
cells treated with Lat A were capable of colony formation (Fig.
1F) and showed a modest, but convincing, increase in cell
number, compared with DMSO-treated wild-type cells (Fig.
1G). However, clp1∆ cells were unable to form colonies on Lat
A-containing plates and did not show a significant increase in
cell number upon Lat A treatment (Fig. 1F and G).
Taken together, these results clearly demonstrate that upon
low-dose Lat A treatment, wild-type cells are competent to
form septa as well as inhibited in progression through further
nuclear cycles. In contrast, clp1∆ mutants are unable to form
septa and proceed through subsequent nuclear cycles without
delay. The ability of wild-type, but not clp1∆ cells, to delay
nuclear division upon Lat A treatment following failed
cytokinesis was also established by imaging individual
cells expressing a histone-GFP fusion (see Movies 1-4,
http://jcs.biologists.org/supplemental/).
Because clp1∆ mutants are sensitive to perturbations to Factin, myosin II heavy and light chains, the PCH domain
protein Cdc15p, cell wall enzymes and potential regulators of
secretion, it is unlikely that Clp1p acts in a redundant
cytokinetic pathway with all these components. We concluded
that an important physiological role for Clp1p was in
promoting cell viability upon delays in cytokinesis caused
by a variety of perturbations that affected the cytokinetic
machinery.
Clp1p has a well-defined role in regulating mitotic entry
(Trautmann et al., 2001; Cueille et al., 2001) (Fig. 1C). It
was thus possible that Clp1p promoted the completion of
cytokinesis indirectly through mediating an interphase (G2)
arrest. If this were the case (i.e. if Clp1p was required solely
for nuclear cycle delay and not for the physical assembly of
division septa) then an artificially induced cell cycle block
should be sufficient to allow completion of cytokinesis in low
dose Lat A treated clp1∆ cells. Conversely, if Clp1p possessed
additional function(s) required for division septum assembly,
then an artificially induced cell cycle block would be
insufficient to allow completion of cytokinesis.
To distinguish between these two possibilities we utilized a
cdc25-22 mutant to arrest cell cycle progression in G2
independently of Clp1p (Berry and Gould, 1996).
Logarithmically growing cdc25-22 and cdc25-22 clp1∆ cells
were shifted to the restrictive temperature of 36°C to
synchronize cells in late G2. Cells were then released to the
permissive temperature of 25°C to allow entry into the first
mitosis. During the shift-down, cells were treated with a low
dose of Lat A to perturb the actomyosin ring and then shifted
back to 36°C in order to block entry into the subsequent mitosis
(Fig. 2A). Using this strategy we were able to assay the
septum-forming ability of clp1∆ mutants in the presence of low
doses of Lat A under conditions where nuclear cycle
progression was blocked by a means that did not require Clp1p
itself. Intriguingly, even though the majority of cdc25-22 cells
were able to form improper but complete septa, cdc25-22
3900
Journal of Cell Science 117 (17)
Cytokinesis checkpoint in fission yeast
Fig. 1. Clp1p is essential when cell division structures are mildly
perturbed and ensures the completion of cytokinesis. (A) Cells of the
indicated genotype were streaked to YES plates and assayed for
colony formation after 3 days at 30°C (left) and 32°C (right).
(B) Cells of indicated genotype were cultured to exponential growth
phase at 24°C and then shifted to 32°C for 5 hours, fixed and then
stained with aniline blue and DAPI to visualize cell wall/septa and
nuclei respectively. Septa are indicated with arrowheads. Scale bar
10 µm. (C) Septum formation in group I cytokinesis mutants at semipermissive temperatures in the presence or absence of Clp1p. Cells
of the indicated genotype were cultured to exponential growth phase
at 24°C, shifted to 32°C for 5 hours, fixed and then stained with
aniline blue and DAPI to visualize cell wall/septa and nuclei
respectively. (D) Wild-type and clp1∆ cells were cultured to
exponential growth phase at 24°C, synchronized in early G2 by
centrifugal elutriation and then treated with a low dose (0.2 µM) of
Lat A or DMSO (solvent control) and cultured at 32°C. Cells were
subsequently fixed at 30 minute intervals and stained with DAPI
(nuclei) and aniline blue (cell wall/septa). (E) Wild-type and clp1∆
cells treated as in Fig. 1D (at t=240 minutes) and stained with both
DAPI (nuclei) and aniline blue (cell wall/septa). Imperfect, but
functional, septa are indicated with arrowheads. Scale bar 10 µm.
(F) Tenfold serial dilutions of wild-type and clp1∆ cells grown on
YES plates containing either 0.25 µM Lat A or DMSO. Images were
captured after 3 days growth at 32°C. (G) Wild-type and clp1∆ cells
were cultured to exponential growth phase at 24°C, synchronized in
early G2 by centrifugal elutriation and then treated with a low dose
(0.2 µM) of Lat A or DMSO (solvent control) and cultured at 32°C.
Cell number was determined using a haemocytometer.
3901
clp1∆ cells were not (Fig. 2B,C). Double mutants generated
predominantly binucleate cells with non-functional spot-like
deposits of septum material. The functionality of septa in
cdc25-22 versus clp1∆ cdc25-22 cells was confirmed by 3D
reconstructions of de-convolved Z-stack images. While cdc2522 cells generally formed mis-shapen, but complete septa that
clearly bisected the cell into two independent compartments,
clp1∆ cdc25-22 cells formed fragmented, incomplete septa that
were unable to divide the cell into two (Fig. 2D). Thus, in
addition to its role in delaying entry into mitosis, Clp1p must
also possess other function(s) that contribute to successful cell
division when progression through cytokinesis is delayed.
Clp1p-dependent maintenance of the actomyosin ring
upon perturbation of cytokinesis
In fission yeast, F-actin is detected in patch-structures
concentrated at the cell tips during interphase and in a ring at
the medial region of the cell in mitotic and post-mitotic cells
undergoing cytokinesis (Marks and Hyams, 1985).
Intriguingly, F-actin is also retained in the medial ring in a high
proportion of cps1-191 single mutants even though arrested
cps1-191 cells contain two G2 nuclei (Liu et al., 2000; Le Goff
et al., 1999b). We therefore considered the possibility that
Clp1p might also be involved in actively maintaining the
actomyosin ring to ensure completion of cytokinesis.
Consistent with this possibility, we found that the maintenance
of medial actomyosin rings in cps1-191 mutants depended
on Clp1p function (Fig. 3A).
Fig. 2. Nuclear cycle arrest is insufficient to allow completion
of cytokinesis upon perturbation of the cell division machinery.
(A) cdc25-22 and clp1∆ cdc25-22 cells were grown to early log
phase at 24°C and then shifted to 36°C for three hours to arrest
cells at the G2/M transition. Cells were subsequently shifted
down to 24°C for 30 minutes, treated with a low dose of Lat A
(0.2 µM) (t=0) to perturb the actomyosin ring and then shifted
back to 36°C at t=30 minutes in the continuing presence of the
drug. (B) Quantitative data for cdc25-22 and clp1∆cdc25-22
cells treated as in Fig. 2A and then fixed with methanol, washed
twice with PBS, and stained with DAPI (nuclei) and aniline
blue (septa) at the indicated time points. Septa were classified
into three groups: normal (similar to septa formed in wild type
cells during logarithmic growth); imperfect and complete
(functional septa that appeared thicker and more disorganized
but bisected the cell); and spotty and incomplete (nonfunctional deposits of septal material that failed to form a linear
structure across the width of the cell). (C) cdc25-22 and
clp1∆cdc25-22 cells treated as in Fig.
2A (at t=4 hours) and stained with both
DAPI (nuclei) and aniline blue (cell
wall/septa). Imperfect but functional,
septa are indicated with arrowheads.
(D) cdc25-22 and clp1∆cdc25-22 cells
treated as in Fig. 2A (at t=4 hours) and
stained with aniline blue (cell
wall/septa). Z-series were obtained and
deconvolved as described in Materials
and Methods. Max projections of the
entire cell are shown to the left of each
panel, whereas three alternate views of
3D reconstructions of septa are shown
to the right.
3902
Journal of Cell Science 117 (17)
Fig. 3. Clp1p is important for the maintenance of the actomyosin ring at the medial region of the cell. (A) cps1-191 and clp1∆cps1-191 were
grown to exponential phase at 24°C, shifted to 36°C for four hours and stained with rhodamine-conjugated phalloidin to determine the
distribution of actin. ‘Medial’ refers to the localization of actin in a medial actomyosin ring structure. ‘Tip’ refers to the localization of actin
patches to the cell ends. (B) Wild-type and clp1∆ cells were cultured to exponential growth phase at 24°C, synchronized in early G2 by
centrifugal elutriation and then treated with a low dose (0.2 µM) of Lat A or DMSO (solvent control). Cells were subsequently fixed at 30minute intervals and stained with DAPI (nuclei) and ALEXA-488 conjugated phalloidin (actin). The graph shows the percent cells with
actomyosin rings. (C) Images of cells treated as in B.
Cytokinesis checkpoint in fission yeast
To address the role of Clp1p in maintenance of actomyosin
rings more rigorously, we studied the localization of F-actin in
wild-type cells and clp1∆ cells treated with a low dose of Lat
A, which we have shown leads to a significant delay in
cytokinesis. We predicted that F-actin rings would be
maintained for a long period of time in wild-type cells treated
with a low dose of Lat A, whereas rings would not be
maintained in clp1∆ cells. Wild-type and clp1∆ cells were
synchronized in early G2, treated with low doses of Lat A, and
were then fixed and stained at 30 minute intervals to determine
the proportion of cells with medial F-actin structures. clp1∆
and wild-type cells were able to form medial rings upon entry
into the first mitosis (~30% and 40% of cells, respectively),
suggesting that clp1∆ cells were fully capable of actomyosin
ring assembly in the presence of low doses of Lat A. The
kinetics of ring assembly upon entry into the first mitosis was
also similar in cells treated with Lat A or DMSO. Interestingly,
wild-type cells could maintain medial F-actin ring structures
for the duration of the experiment (Fig. 3B,C) and were able
to assemble improper but complete division septa indicative of
successful ring constriction (Fig. 1D,E). The appearance of
rings ranged from normal to malformed. They contained
myosin II (data not shown) and were predominantly in the
medial region of the cell as a broader structure. Unlike wildtype cells, medial F-actin rings in clp1∆ cells disassembled
upon mitotic exit leading to the formation of unseptated cells
with two nuclei. Upon failed cytokinesis in the clp1∆ mutant
nuclei were observed to cluster (Fig. 3C, marked with
arrowheads), as typically observed in SIN mutants that fail in
cytokinesis (Hagan and Yanagida, 1997). In contrast, the nuclei
in wild-type cells treated with Lat A do not cluster (Fig. 3C,
marked with arrows), presumably due to the maintenance of
the underlying post-anaphase array of microtubules (M.M.,
unpublished) as has been reported for other group I mutants
(Pardo and Nurse, 2003; Liu et al., 2002). The synchronous
culture experiment established that the actomyosin ring was
maintained for a prolonged period of time in the presence of
Clp1p but not in its absence. We concluded that Clp1p function
was important to maintain the actomyosin ring at the medial
region of the cell until cytokinesis was complete, in particular
under adverse conditions.
We have shown that the actomyosin ring is not maintained
in clp1∆ cells upon perturbation of the cytokinetic machinery.
We therefore investigated the dynamics of the actomyosin ring
upon low dose Lat A treatment to gain insight into the
mechanism of actomyosin ring maintenance in wild-type cells
and its collapse in clp1∆ cells. We utilized wild-type or clp1∆
mutant cells expressing a GFP-tagged version of the
actomyosin ring component Rlc1p (Naqvi et al., 2000; Le Goff
et al., 2000) and performed time-lapse imaging of these cells
treated with either DMSO (control) or a low dose of Lat A. At
least 10 cells with medial rings with well-separated nuclei
were typically imaged for each experiment. Dynamics of
actomyosin ring constriction and progression through
cytokinesis in DMSO treated clp1∆ cells was comparable to
that in wild-type cells and the process of ring constriction and
septum assembly typically lasted between 25-35 minutes (Fig.
4). As shown earlier (Fig. 3B,C), clp1∆ cells treated with a low
dose of Lat A were capable of assembling actomyosin rings.
However, these rings did not constrict upon completion of
mitosis but fragmented leading to the loss of the actomyosin
3903
ring (Fig. 4A; marked with an arrow). We then imaged wildtype cells treated with a low dose of Lat A to study actomyosin
ring dynamics in these cells (Fig. 4A). Interestingly, the
actomyosin ring was maintained for prolonged periods
between 40-80 minutes and eventually underwent slow
constriction (60 and 97 minutes in Fig. 4A) and division
septum assembly leading to cell division (arrowhead in Fig.
4A).
To observe ring structure more closely we performed similar
experiments in which 3D reconstructions of de-convolved Zstack images were examined. These results were similar to our
previous time lapse microscopy results and clearly showed that
rings in clp1∆ cells fragmented in response to Lat A whereas
rings in wild-type cells were stabilized to a sufficient degree to
allow constriction, albeit over a much longer time frame than
DMSO controls (Fig. 4B). Thus, whereas in wild-type cells,
where the actomyosin ring is maintained for a long period of
time and its constriction is slowed down but completed upon
exposure to a low dose of Lat A, the rings disassemble under
a similar regime in clp1∆ cells.
Previous studies have shown that SIN mutants assemble
actomyosin rings that are not maintained upon completion of
mitosis, leading to defects in septation (Gould and Simanis,
1997; Guertin et al., 2002; Balasubramanian et al., 1998). We
considered the possibility that the dynamics of the actomyosin
ring in SIN mutants might phenocopy the actomyosin ring loss
phenotype of clp1∆ cells treated with a low dose of Lat A. To
address this question, sid1-239 and sid2-250 cells (Fig. 4; data
not shown for spg1-106 and cdc14-118) expressing Rlc1p-GFP
were shifted to the restrictive temperature and the dynamics of
the actomyosin ring monitored. SIN mutants were capable of
assembling medial actomyosin rings as previously shown in
several studies (Gould and Simanis, 1997; Guertin et al., 2002;
Balasubramanian et al., 1998). Interestingly, these actomyosin
rings disassembled upon completion of mitosis (marked with
arrows in Fig. 4A) in a manner similar to that observed in lowdose Lat A treated clp1∆ cells. Furthermore, sid1-239 clp1∆
cells shifted to semi-permissive temperatures in the presence
of DMSO or Lat A showed a ring disassembly phenotype
similar to that shown by sid1-239 single mutants at the
restrictive temperature, suggesting that Sid1p and Clp1p
function in a common pathway rather than in overlapping
additive pathways (Fig. 4B). Based on these studies we
conclude that the SIN is important for actomyosin ring
maintenance during every cell cycle, whereas Clp1p is
important for actomyosin ring maintenance only if the cell
division machinery is perturbed.
Active SIN signaling greatly bypasses the need for
Clp1p function upon perturbation of the cell division
machinery
The SIN plays an important role in the G2 delay following
cytokinesis failure (Gould and Simanis, 1997; Feierbach and
Chang, 2001). Thus, we wanted to test whether the SIN, like
Clp1p, was essential for cell viability when cytokinesis was
slowed down. Because the SIN is essential we used a SIN
mutant, cdc14-118, that was partially active, but viable, at
30°C. We found that myo2-E1 cdc14-118 cells resembled
myo2-E1 clp1∆ mutants (Fig. 1A,B) in that these cells were
inviable at 30°C, whereas both single mutants were viable at
3904
Journal of Cell Science 117 (17)
Cytokinesis checkpoint in fission yeast
Fig. 4. Dynamics of Clp1p-dependent actomyosin ring maintenance
upon perturbation of cytokinetic machinery. (A) Cells of the
indicated genotypes expressing Rlc1p-GFP were imaged under
conditions shown on the left-hand side. The numbers on the top right
of each frame indicates the time in minutes. Note that whereas the
Rlc1p-GFp rings are maintained for a prolonged period of time,
leading to the accomplishment of division septum assembly in wildtype cells upon treatment with a low dose of Lat A (0.2 µM), these
rings fragment in similarly treated clp1∆ cells. Note also that SIN
mutants display a phenotype similar to that observed in clp1∆ cells
treated with a low dose of Lat A. (B) 3D-reconstructions of Rlc1pGFP expressing cells of the indicated genotype treated with DMSO
or 0.2 µM Lat A. Time (t) is indicated in minutes. Wild-type, clp1∆,
and clp1∆ sid1-239 cells were imaged at 32°C. sid1-239 cells were
imaged at 36°C.
this temperature. Examination of the double mutants showed
that they were multinucleate and were incapable of assembling
division septa at 30°C, whereas the single mutants were largely
unaffected (Fig. 5A,B). Furthermore, all SIN single mutants
treated with low doses of Lat A under permissive and semipermissive conditions accumulated multiple nuclei and failed
to septate similar to clp1∆ mutants (data not shown). Given that
SIN mutants display a phenotype similar to that displayed by
clp1∆ mutants upon mild damage to the cell division apparatus,
and that ectopic SIN activation leads to septation in interphase
cells, we reasoned that SIN hyper-activation might rescue the
checkpoint defect in clp1∆ cells. We therefore examined if
ectopic activation of the SIN by mutation of cdc16 (encodes a
GAP for the Spg1p GTPase) (Minet et al., 1979; Fankhauser
et al., 1993; Furge et al., 1998) allowed clp1∆ cells treated with
low doses of Lat A to complete division septum assembly and
arrest with two nuclei. Cells of the genotypes cdc16-116,
clp1∆, and cdc16-116 clp1∆ were treated with a low dose of
Lat A and shifted to the restrictive temperature of 36°C for four
hours to allow inactivation of the temperature-sensitive Cdc16116p (Fig. 5C,D). Interestingly, we observed a high proportion
of clp1∆ cdc16-116 cells with two nuclei and an improper but
complete division septum (marked with arrowheads in Fig.
5C). This was in contrast to clp1∆ single mutants where only
3% of cells displayed such a phenotype. These observations
suggested that ectopic activation of the SIN cascade
significantly rescues the G2 arrest and septation defects of
clp1∆ cells upon perturbation of the cytokinetic machinery and
suggests that a normal role for Clp1p is to prolong SIN
signaling to allow completion of cytokinesis. Similar results
were obtained when the cytokinesis checkpoint was activated
using other means, such as using the cps1-191 mutant (data not
shown).
To further understand the mechanism by which Clp1p
influenced SIN function, we assayed the localization of SIN
components in wild-type and clp1∆ cells exposed to low doses
of Lat A. The SIN is composed of a group of spindle pole body
localized proteins. Of these, Cdc7p, Sid1p, and Cdc14p
localize to one SPB in late mitosis and remain there until the
completion of cytokinesis. Previous studies have proposed that
the localization of these proteins to a single SPB signifies
active SIN signaling (reviewed in McCollum and Gould,
2001). We therefore assessed the kinetics of localization of
Cdc7p and Sid1p in wild-type and clp1∆ cells treated with Lat
A. Synchronous populations of wild-type and clp1∆ cells
3905
expressing GFP-Sid1p or Cdc7p-GFP were generated by
centrifugal elutriation and released into growth medium
containing Lat A. As in previous experiments, wild-type and
clp1∆ cells entered mitosis with roughly comparable kinetics.
Sid1p-GFP and Cdc7p-GFP were also detected on one spindle
pole body with relatively similar kinetics (Fig. 6A). However,
whereas Sid1p-GFP and Cdc7p-GFP were retained at the SPB
for prolonged periods in wild type cells, until completion of
cytokinesis, Sid1p-GFP and Cdc7p-GFP were undetectable in
clp1∆ cells treated with Lat A (Fig. 6A). Thus, we conclude
that Clp1p is not required for the initial localization of Sid1p
and Cdc7p to one SPB during mitosis, but is required to
maintain them at the SPB upon cytokinesis delay in the
presence of Lat A, to allow completion of cell division.
We then studied the role of SIN in Clp1p function. Previous
studies have shown that Clp1p is present in the nucleolus in
interphase cells and is released from the nucleolus to the
cytoplasm in cells undergoing cytokinesis (Trautmann et al.,
2001; Cueille et al., 2001). In addition, Clp1p is also detected
at the SPB, mitotic spindle, and the actomyosin ring. We have
shown that weak cytokinetic defects caused by faults in various
components of the cell division apparatus result in lethality to
SIN mutants even under permissive temperature conditions,
suggesting a role for SIN in ‘cytokinesis checkpoint’ function.
We therefore studied the localization of Clp1p-GFP in wildtype and cdc14-118 mutants (grown at permissive temperature)
in the presence and absence of Lat A. In wild-type and cdc14118 mutants treated with DMSO, Clp1p-GFP was in the
nucleolus of interphase cells and in the cytoplasm of mitotic
and cytokinetic cells. Interestingly, whereas Clp1p-GFP was
retained in the cytoplasm of wild-type cells treated with Lat A,
Clp1p-GFP readily relocalized to the nucleolus in cdc14-118
cells treated with Lat A (Fig. 6B). We therefore conclude that
Clp1p-GFP maintenance in the cytoplasm in response to
cytokinesis failure requires functional SIN.
Discussion
In this study we have provided evidence for, and described the
physiological role of Clp1p in ensuring cell viability upon mild
perturbation of the cell division machinery. Based on this
evidence we propose a model in which Clp1p, together with
its effector, the SIN, form an integral part of a cytokinesis
checkpoint that ensures damaged cell division structures are
maintained and/or reformed in order to allow the completion
of cytokinesis.
Clp1p, a bona fide checkpoint protein, and its
physiological role
We propose that aspects of cytokinesis, such as actomyosin ring
function, cell wall assembly, and possibly secretion are
monitored by the cytokinesis checkpoint, through a mechanism
requiring Clp1p and the SIN. This is demonstrated by the
dramatic cytokinetic failure of clp1 and SIN mutants (under
semi-permissive conditions) when the cell division machinery is
perturbed through the use of a variety of cytokinesis mutants as
well as through the use of cytoskeletal inhibitors. The mutants
studied include those that are defective in actomyosin ring
assembly, division septum assembly, as well as a type V myosin
mutant that is likely involved in vesicle targeting. The fact that
3906
Journal of Cell Science 117 (17)
Fig. 5. SIN mutants are hypersensitive to mild perturbations of the cell division machinery whereas ectopic activation of the SIN compensates
for the loss of Clp1p. (AI) cdc14-118, myo2-E1, and myo2-E1 cdc14-118 mutants were streaked to YES plates and assayed for colony
formation after 3 days at the semi-permissive temperature of 30°C. (AII, III and IV) cdc14-118, myo2-E1 and myo2-E1 cdc14-118 cells were
grown at 24°C to early-log phase, shifted to 30°C for six hours, and then fixed and stained with DAPI and aniline blue to visualize nuclei and
division septa, respectively. Scale bar 10 µm. (B) Quantitative data for cells in AII-IV. (C) Cells of the indicated genotypes were grown to earlylog phase at 24°C, shifted to 36°C, and then treated with 0.2 µM of Lat A or DMSO (solvent control) for 5 hours. Cells were subsequently fixed
and stained with DAPI and aniline blue to visualize nuclei and division septa respectively. Scale bar 10 µm. (D) Quantitative data for cells
treated with Lat A in C.
Clp1p is a component of the actomyosin ring (Trautmann et al.,
2001; Cueille et al., 2001) makes it an attractive candidate for
monitoring the progression/completion of cytokinesis. However,
it is equally likely that Clp1p is a downstream effector that
responds to cues initiated upon a failure of cytokinesis.
It is also interesting to note the parallels between Clp1p and
other previously described checkpoint proteins, such as Mad2p
and the budding yeast Rad9p (He et al., 1997; Weinert and
Hartwell, 1988). All three of these proteins are normally nonessential, but do play a critical role in ensuring viability upon
perturbation of actin/cell wall, microtubular machinery and DNA
integrity, respectively. The fact that cells defective for Clp1p are
sensitive to a variety of perturbations to the cell division
machinery is also consistent with a role for Clp1p in monitoring
several aspects of cytokinesis. This is akin to the sensitivity of,
for example, rad9 mutants to a variety of DNA damaging agents.
What is the normal function of this checkpoint? The
checkpoint might be involved in allowing a period of time to
correct minor defects in the assembly of the cell division
apparatus due to temperature shifts, or possibly toxins
(equivalent to cytochalasin A, latrunculin A, pneumocandins
and echinocandins) (Cooper, 1987; Denning, 1997;
Georgopapadakou, 1998; Spector et al., 1989; Ayscough et al.,
1997) secreted by other organisms that destroy the actomyosin
ring, cell wall biosynthetic machinery, or secretory machinery.
The fact that a low percentage of clp1– cells display cytokinesis
defects (Cuielle et al., 2001; Trautmann et al., 2001) indicates
that processes, such as actomyosin ring assembly and cell wall
secretion in normal wild-type cells are also prone to errors that
might be corrected by the cytokinesis checkpoint.
Repair of the cell division apparatus and completion of
division septum assembly in response to activation of
the cytokinesis checkpoint
What are the responses to cytokinesis checkpoint activation?
Cytokinesis checkpoint in fission yeast
3907
Fig. 6. (A) Wild-type and clp1∆ cells carrying
integrated copies of Sid1p-GFP and Cdc7p-GFP were
cultured to exponential growth phase at 24°C,
synchronized in early G2 by centrifugal elutriation and
then treated with a low dose (0.2 µM) of Lat A or
DMSO (solvent control) and cultured at 32°C. Cells
were subsequently fixed at 30 minute intervals, stained
with antibodies against GFP (see Materials and
Methods), and scored for localization of Sid1p-GFP
(left) and Cdc7p-GFP (right) to the SPB. (B) Wild-type
and cdc14-118 cells carrying an integrated copy of
Clp1p-GFP were treated with 0.2 µM Lat A or DMSO,
shifted to 32°C for 3.5 hours, fixed and then stained
with DAPI. Clp1p-GFP localization was monitored
using GFP autofluorescence.
In this and previous studies, it has been shown that the
checkpoint prevents the two interphase nuclei following failed
cytokinesis from entering mitosis (Trautmann et al., 2001;
Cueille et al., 2001). We have shown the activation of the
checkpoint actively maintains physical structures important for
cell division until cytokinesis is completed (Figs 3, 4). We have
also shown that in cells lacking Clp1p, a G2 delay provided
using a cdc25-22 mutation is insufficient to allow completion
of division septum assembly. This result establishes that Clp1p
has independent roles in the physical maintenance of the
actomyosin ring and in providing a G2 delay upon perturbation
of the cell division apparatus. These data also establish that
negative regulation of Cdc2p kinase function is not the sole
function of Clp1p, but that Clp1p also functions (via SIN,
discussed in a later section) to maintain the actomyosin ring in
a Cdc2p-independent manner.
While the molecular role of Clp1p in re-establishing cell
division structures is unclear, it appears that a major function
of Clp1p is to keep the SIN active in order to effect G2 arrest
and the reassembly of actomyosin rings and completion of
assembly of division septa. This and previous studies have
shown that Cdc7p and Sid1p are retained at one SPB in a
Clp1p-dependent manner in wild-type cells treated with Lat A
and in cps1-191 mutants at the restrictive
temperature (Trautmann et al., 2001) suggesting a
role for Clp1p in prolonging the duration of SIN
signaling to effect completion of cytokinesis. The
suggestion that Clp1p functions by prolonging the
duration of SIN signaling is also consistent with
the similar phenotypes observed (summarized in
Fig. 7A) in SIN mutants at the fully restrictive
temperature, as well as clp1∆ cells in which the
cytokinetic machinery has been perturbed through
treatment with Lat A. Interestingly, the
localization of Clp1p itself appears to depend on
SIN function, given that Clp1p is not maintained
in the cytoplasm in cdc14-118 (a SIN mutant)
cells treated with Lat A under semi-permissive
conditions, whereas Clp1p is retained in the
cytoplasm of wild-type cells treated with Lat A
(Fig. 6). Thus, we propose that Clp1p and SIN
function in a positive-feedback loop in which the
localization SIN depends on Clp1p and the
maintenance of Clp1p in the cytoplasm depends
on SIN function (Fig. 7B). We have also shown
that the requirement for Clp1p in G2 delay and in reestablishment and maintenance of cell division structures is
significantly bypassed by ectopic activation of the SIN (Fig. 5).
Thus, although SIN and Clp1p function in a positive feedback
loop, active SIN alone is largely sufficient to allow completion
of cytokinesis and maintain G2 arrest. Previous studies have
shown that ectopic activation of the SIN results in repeated
rounds of septation in interphase arrested cells (Schmidt et al.,
1997; Fankhauser et al., 1993; Balasubramanian et al., 1998;
Minet et al., 1979). The ability of cells to complete septation
in a SIN and Clp1p-dependent manner from interphase, upon
perturbation of the cell division apparatus, might provide a
physiological explanation for the observed ability of ectopic
SIN signaling to promote septation in interphase arrested cells
(Minet et al., 1979; Fankhauser et al., 1993; Schmidt et al.,
1997).
It is currently unclear whether Clp1p acts at the level of
Byr4p-Cdc16p complex, Plo1p, or at the level of Spg1p, all of
which can be modulated to maintain an active SIN cascade
(Minet et al., 1979; Furge et al., 1998; Schmidt et al., 1997;
Balasubramanian et al., 1998). Given that the Cdc14p family
of phosphatases has been shown to reverse Cdc2p/Cdk1pphosphorylation on several of its substrates (Visintin et al., 1998;
3908
Journal of Cell Science 117 (17)
Fig. 7. Summary and Model. (A) Summary of cellular
behavior in differing genotypes and growth conditions.
Actin patches and rings are shown in red. Nuclei are shown
as black circles. Cdc7p localization to the spindle pole body
(a marker of active SIN) is shown schematically as a blue
circle. Wild-type or clp1∆ cells under normal growth
conditions (column 1); wild-type or clp1∆ cells upon
treatment with low doses of Lat A (columns 2 and 3,
respectively); cps1-191 and clp1∆ cps1-191 cells at the
restrictive temperature of 36°C (columns 4 and 5,
respectively); a SIN mutant at the restrictive temperature of
36°C (column 6). (B) Model. Upon perturbation of the
cytokinesis machinery Clp1p and the SIN function in a
positive feedback loop to maintain the integrity of the
actomyosin ring (see Discussion).
Ubersax et al., 2003), Clp1p might also regulate SIN through
other mechanisms not involving known components of the SIN.
SIN components: essential proteins important for the
cytokinesis checkpoint
Of particular interest is the similarity between the phenotype of
SIN mutants at the fully restrictive temperature, and low dose
Lat A treated clp1∆ cells (Fig. 4). In both cases, the primary
defect is a failure to maintain the integrity of the ring leading
ultimately to its disassembly. Because SIN genes are essential,
and clp1 non-essential, the simplest interpretation taking all data
together is that Clp1p function is required for ring maintenance
only upon perturbation of the cell division machinery while SIN
function is required for maintenance of the actomyosin ring at
the end of every cell cycle during ring constriction and septation.
It is also possible that SIN has other essential functions in
cytokinesis, because delivery and assembly of Cps1p, an integral
membrane protein important for division septum assembly is
abrogated in SIN mutants (Liu et al., 2002; Cortes et al., 2002).
Does a similar checkpoint mechanism operate in other
organisms?
Several lines of evidence suggest the existence of similar arrest
mechanisms in mammalian cells upon perturbations to the
cytokinetic machinery. Animal cells that exit mitosis while
cytokinesis is blocked remain arrested in a telophase like state
with persistent cell division structures. Furthermore, these
cells can initiate cytokinesis when the cytokinesis block is
removed (Martineau et al., 1995; Canman et al., 2000). In
addition, mammalian cells impaired for synthesis of
phosphotidylethanolamine are unable to carry out cytokinesis
and arrest with a stable actomyosin ring and interphase nuclei
(Emoto and Umeda, 2000). Finally, mammalian cells treated
with a myosin II ATPase inhibitor arrest as binucleate cells
with intact actomyosin rings and the underlying microtubular
network as in fission yeast cells (Straight et al., 2003; Pardo
and Nurse, 2003). Presently it is unclear how this arrest is
mediated in metazoan cells. However, proteins similar to Clp1p
and SIN components (such as Mob1p and Cdc11p) have been
identified in several eukaryotes including animal cells (Bardin
and Amon, 2001; Pereira and Schiebel, 2001; Bembenek and
Yu, 2001; Gruneberg et al., 2002; Moreno et al., 2001;
Gromley et al., 2003), raising the possibility that similar
molecules might function in cytokinesis checkpoint-like
processes in a variety of eukaryotic cell types.
In summary, we have demonstrated that the cytokinesis
checkpoint mechanism in fission yeast is essential for cell
viability in conditions where the cell division machinery is
Cytokinesis checkpoint in fission yeast
mildly perturbed. The cytokinesis checkpoint responds by
providing an interphase arrest, and by promoting the
reassembly of cell division structures, such as the actomyosin
ring and the division septum. These two responses thereby
allow the completion of cytokinesis before the subsequent
mitosis. These mechanisms operate to ensure that a mother cell
divides to produce two viable daughter cells whose ploidy is
identical to that of the mother. In future it will be important to
identify the nature of the signal(s) that are monitored by the
cytokinesis checkpoint, the detailed mechanism by which
actomyosin rings and possibly other cell division structures are
reassembled, and the molecular mechanism leading to G2
arrest.
We wish to thank all members of the yeast and fungal biology
laboratories at the Temasek Life Sciences Laboratories for their
encouragement, advice and critical reading of the manuscript and
Michael Glotzer and Uttam Surana for critical comments and
discussion. This work was supported by research funds from the
Temasek Life Sciences Laboratory. J.K. is a recipient of the Singapore
Millennium Foundation Post-Doctoral Fellowship. D.M. is supported
by grants from the NIH, USA.
References
Ayscough, K. R., Stryker, J., Pokala, N., Sanders, M., Crews, P. and
Drubin, D. G. (1997). High rates of actin filament turnover in budding yeast
and roles for actin in establishment and maintenance of cell polarity revealed
using the actin inhibitor latrunculin-A. J. Cell Biol. 137, 399-416.
Balasubramanian, M. K., McCollum, D. and Gould, K. L. (1997).
Cytokinesis in fission yeast Schizosaccharomyces pombe. Methods Enzymol.
283, 494-506.
Balasubramanian, M. K., McCollum, D., Chang, L., Wong, K. C., Naqvi,
N. I., He, X., Sazer, S. and Gould, K. L. (1998). Isolation and
characterization of new fission yeast cytokinesis mutants. Genetics 149,
1265-1275.
Bardin, A. J. and Amon, A. (2001). Men and sin: what’s the difference? Nat.
Rev. Mol. Cell. Biol. 2, 815-826.
Bembenek, J. and Yu, H. (2001). Regulation of the anaphase-promoting
complex by the dual specificity phosphatase human Cdc14a. J. Biol. Chem.
276, 48237-48242.
Berry, L. D. and Gould, K. L. (1996). Regulation of Cdc2 activity by
phosphorylation at T14/Y15. Prog. Cell Cycle Res. 2, 99-105.
Bezanilla, M., Forsburg, S. L. and Pollard, T. D. (1997). Identification of a
second myosin-II in Schizosaccharomyces pombe: Myp2p is conditionally
required for cytokinesis. Mol. Biol. Cell 8, 2693-2705.
Canman, J. C., Hoffman, D. B. and Salmon, E. D. (2000). The role of preand post-anaphase microtubules in the cytokinesis phase of the cell cycle.
Curr. Biol. 10, 611-614.
Chang, F., Woollard, A. and Nurse, P. (1996). Isolation and characterization
of fission yeast mutants defective in the assembly and placement of the
contractile actin ring. J. Cell Sci. 109, 131-142.
Cooper, J. A. (1987). Effects of cytochalasin and phalloidin on actin. J. Cell
Biol. 105, 1473-1478.
Cortes, J. C., Ishiguro, J., Duran, A. and Ribas, J. C. (2002). Localization
of the (1,3)beta-D-glucan synthase catalytic subunit homologue
Bgs1p/Cps1p from fission yeast suggests that it is involved in septation,
polarized growth, mating, spore wall formation and spore germination. J.
Cell Sci. 115, 4081-4096.
Cueille, N., Salimova, E., Esteban, V., Blanco, M., Moreno, S., Bueno, A.
and Simanis, V. (2001). Flp1, a fission yeast orthologue of the S. cerevisiae
cdc14 gene, is not required for cyclin degradation or rum1p stabilisation at
the end of mitosis. J. Cell Sci. 114, 2649-2664.
Denning, D. W. (1997). Echinocandins and pneumocandins-a new antifungal
class with a novel mode of action. J. Antimicrob. Chemother. 40, 611-614.
Emoto, K. and Umeda, M. (2000). An essential role for a membrane lipid in
cytokinesis. Regulation of contractile ring disassembly by redistribution of
phosphatidylethanolamine. J. Cell Biol. 149, 1215-1224.
Eng, K., Naqvi, N. I., Wong, K. C. and Balasubramanian, M. (1998).
3909
Rng2p, a protein required for cytokinesis in fission yeast, is a component of
the actomyosin ring and the spindle pole body. Curr. Biol. 8, 611-621.
Fankhauser, C., Marks, J., Reymond, A. and Simanis, V. (1993). The S.
pombe cdc16 gene is required both for maintenance of p34cdc2 kinase
activity and regulation of septum formation: a link between mitosis and
cytokinesis? EMBO J. 12, 2697-2704.
Fankhauser, C., Reymond, A., Cerutti, L., Utzig, S., Hofmann, K. and
Simanis, V. (1995). The S. pombe cdc15 gene is a key element in the
reorganization of F-actin at mitosis. Cell 82, 435-444.
Feierbach, B. and Chang, F. (2001). Cytokinesis and the contractile ring in
fission yeast. Curr. Opin. Microbiol. 4, 713-719.
Field, C., Li, R. and Oegema, K. (1999). Cytokinesis in eukaryotes: a
mechanistic comparison. Curr. Opin. Cell Biol. 11, 68-80.
Furge, K. A., Wong, K., Armstrong, J., Balasubramanian, M. and
Albright, C. F. (1998). Byr4 and Cdc16 form a two-component GTPaseactivating protein for the Spg1 GTPase that controls septation in fission
yeast. Curr. Biol. 8, 947-954.
Georgopapadakou, N. H. (1998). Antifungals: mechanism of action and
resistance, established and novel drugs. Curr. Opin. Microbiol. 1, 547-557.
Gould, K. L. and Simanis, V. (1997). The control of septum formation in
fission yeast. Genes Dev. 11, 2939-2951.
Gromley, A., Jurczyk, A., Sillibourne, J., Halilovic, E., Mogensen, M.,
Groisman, I., Blomberg, M. and Doxsey, S. (2003). A novel human
protein of the maternal centriole is required for the final stages of cytokinesis
and entry into S phase. J. Cell Biol. 161, 535-545.
Gruneberg, U., Glotzer, M., Gartner, A. and Nigg, E. A. (2002). The
CeCDC-14 phosphatase is required for cytokinesis in the Caenorhabditis
elegans embryo. J. Cell Biol. 158, 901-914.
Guertin, D., Trautmann, S. and McCollum, D. (2002). Cytokinesis in
eukaryotes. Microbiol. Mol. Biol. Rev. 66, 155-178.
Hagan, I. and Yanagida, M. (1997). Evidence for cell cycle-specific, spindle
pole body-mediated, nuclear positioning in the fission yeast
Schizosaccharomyces pombe. J. Cell Sci. 110, 1851-1866.
He, X., Patterson, T. E. and Sazer, S. (1997). The Schizosaccharomyces
pombe spindle checkpoint protein mad2p blocks anaphase and genetically
interacts with the anaphase-promoting complex. Proc. Natl. Acad. Sci. USA
94, 7965-7970.
Karagiannis, J. and Young, P. G. (2001). Intracellular pH homeostasis during
cell-cycle progression and growth state transition in Schizosaccharomyces
pombe. J. Cell Sci. 114, 2929-2941.
Kelly, T. J. and Brown, G. W. (2000). Regulation of chromosome replication.
Ann. Rev. Biochem. 69, 829-880.
Le Goff, X., Utzig, S. and Simanis, V. (1999a). Controlling septation in fission
yeast: finding the middle, and timing it right. Curr. Genet. 35, 571-584.
Le Goff, X., Woollard, A. and Simanis, V. (1999b). Analysis of the cps1 gene
provides evidence for a septation checkpoint in Schizosaccharomyces
pombe. Mol. Gen. Genet. 262, 163-172.
Le Goff, X., Motegi, F., Salimova, E., Mabuchi, I. and Simanis, V. (2000).
The S. pombe rlc1 gene encodes a putative myosin regulatory light chain
that binds the type II myosins myo3p and myo2p. J. Cell Sci. 113, 41574163.
Liu, J., Wang, H., McCollum, D. and Balasubramanian, M. K. (1999).
Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division
septum assembly in Schizosaccharomyces pombe. Genetics 153, 1193-1203.
Liu, J., Wang, H. and Balasubramanian, M. K. (2000). A checkpoint that
monitors cytokinesis in Schizosaccharomyces pombe. J. Cell Sci. 113, 12231230.
Liu, J., Tang, X., Wang, H., Oliferenko, S. and Balasubramanian, M. K.
(2002). The localization of the integral membrane protein Cps1p to the cell
division site is dependent on the actomyosin ring and the septation-inducing
network in Schizosaccharomyces pombe. Mol. Biol. Cell 13, 989-1000.
Marks, J. and Hyams, J. S. (1985). Localization of F-actin through the cell
division cycle of Schizosaccharomyces pombe. Eur. J. Cell Biol. 39, 27-32.
Martineau, S. N., Andreassen, P. R. and Margolis, R. L. (1995). Delay of
HeLa cell cleavage into interphase using dihydrocytochalasin B: retention
of a postmitotic spindle and telophase disc correlates with synchronous
cleavage recovery. J. Cell Biol. 131, 191-205.
McCollum, D. and Gould, K. L. (2001). Timing is everything: regulation of
mitotic exit and cytokinesis by the MEN and SIN. Trends Cell Biol. 11, 8995.
McCollum, D., Balasubramanian, M. K., Pelcher, L. E., Hemmingsen, S.
M. and Gould, K. L. (1995). Schizosaccharomyces pombe cdc4+ gene
encodes a novel EF-hand protein essential for cytokinesis. J. Cell Biol. 130,
651-660.
3910
Journal of Cell Science 117 (17)
Minet, M., Nurse, P., Thuriaux, P. and Mitchison, J. M. (1979).
Uncontrolled septation in a cell division cycle mutant of the fission yeast
Schizosaccharomyces pombe. J. Bacteriol. 147, 440-446.
Moreno, C. S., Lane, W. S. and Pallas, D. C. (2001). A mammalian homolog
of yeast MOB1 is both a member and a putative substrate of striatin familyprotein phosphatase 2A complexes. J. Biol. Chem. 276, 24253-24260.
Moreno, S., Klar, A. and Nurse, P. (1991). Molecular genetic analysis of
fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795-823.
Motegi, F., Nakano, K., Kitayama, C., Yamamoto, M. and Mabuchi, I.
(1997). Identification of Myo3, a second type-II myosin heavy chain in the
fission yeast Schizosaccharomyces pombe. FEBS Lett. 420, 161-166.
Motegi, F., Arai, R. and Mabuchi, I. (2001). Identification of two type V
myosins in fission yeast, one of which functions in polarized cell growth
and moves rapidly in the cell. Mol. Biol. Cell 12, 1367-1380.
Naqvi, N. I., Wong, K., Tang, X. and Balasubramanian, M. K. (2000). Type
II myosin regulatory light chain relieves auto-inhibition of myosin-heavychain function. Nat. Cell Biol. 2, 855-858.
Nurse, P., Thuriaux, P. and Nasmyth, K. (1976). Genetic control of the cell
division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen.
Genet. 146, 167-177.
Nyberg, K. A., Michelson, R., Putnam, C. W. and Weinert, T. A. (2002).
Toward maintaining the genome: DNA damage and replication checkpoints.
Ann. Rev. Genet. 36, 617-656.
Pardo, M. and Nurse, P. (2003). Equatorial retention of the contractile actin
ring by microtubules during cytokinesis. Science 300, 1569-1574.
Pelham, R. J. and Chang, F. (2001). Role of actin polymerization and actin
cables and actin-patch movement in Schizosaccharomyces pombe. Nat. Cell
Biol. 3, 235-244.
Pereira, G. and Schiebel, E. (2001). The role of the yeast spindle pole body
and the mammalian centrosome in regulating late mitotic events. Curr. Opin.
Cell Biol. 13, 762-769.
Rudner, A. D. and Murray, A. (1996). The spindle assembly checkpoint.
Curr. Opin. Cell Biol. 8, 773-780.
Schmidt, S., Sohrmann, M., Hofmann, K., Woollard, A. and Simanis, V.
(1997). The Spg1p GTPase is an essential, dosage-dependent inducer of
septum formation in Schizosaccharomyces pombe. Genes Dev. 11, 15191534.
Spector, I., Shochet, N. R., Blasberger, D. and Kashman, Y. (1989).
Latrunculins-novel marine macrolides that disrupt microfilament
organization and affect cell growth: I. Comparison with cytochalasin D.
Cell. Motil. Cytoskeleton 13, 127-144.
Straight, A. F., Cheung, A., Limouze, J., Chen, I., Westwood, N. J., Sellers,
J. R. and Mitchison, T. J. (2003). Dissecting temporal and spatial control
of cytokinesis with a myosin II Inhibitor. Science 299, 1743-1747.
Trautmann, S., Wolfe, B., Jorgensen, P., Tyers, M., Gould, K. L. and
McCollum, D. (2001). Fission yeast Clp1p phosphatase regulates G2/M
transition and coordination of cytokinesis with cell cycle progression. Curr.
Biol. 11, 931-940.
Ubersax, J. A., Woodbury, E. L., Quang, P. N., Paraz, M., Blethrow, J. D.,
Shah, K., Shokat, K. M. and Morgan, D. O. (2003). Targets of the cyclindependent kinase Cdk1. Nature 425, 859-864.
Visintin, R., Craig, K., Hwang, E. S., Prinz, S., Tyers, M. and Amon, A.
(1998). The phosphatase Cdc14 triggers mitotic exit by reversal of Cdkdependent phosphorylation. Mol. Cell 2, 709-718.
Weinert, T. A. and Hartwell, L. H. (1988). The RAD9 gene controls the cell
cycle response to DNA damage in Saccharomyces cerevisiae. Science 241,
317-322.
Win, T. Z., Gachet, Y., Mulvihill, D. P., Might, K. M. and Hyams, J. S.
(2001). Two type V myosins with non-overlapping functions in the fission
yeast Schizosaccharomyces pombe: Myo52 is concerned with growth
polarity and cytokinesis, Myo51 is a component of the cytokinetic actin ring.
J. Cell Sci. 114, 69-79.
Wong, K. C. Y., D’Souza, V. M., Naqvi, N. I., Motegi, F., Mabuchi, I. and
Balasubramanian, M. K. (2002). Importance of a myosin II-containing
progenitor for actomyosin ring assembly in fission yeast. Curr. Biol. 12, 724729.